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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李財坤(Tsai-Kun Li) | |
| dc.contributor.author | Chia-En Huang | en |
| dc.contributor.author | 黃佳恩 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:24:56Z | - |
| dc.date.available | 2021-02-23 | |
| dc.date.copyright | 2021-02-23 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-11-17 | |
| dc.identifier.citation | 1. Chakraborty P, Huang JTJ, Hiom K. DHX9 helicase promotes R-loop formation in cells with impaired RNA splicing. Nat Commun. 9, 4346 (2018). 2. Jaber S, Toufektchan E, Lejour V, Bardot B, Toledo F. p53 downregulates the Fanconi anaemia DNA repair pathway. Nat Commun. 7, 11091 (2016). 3. Rodríguez-Cortez VC, Martínez-Redondo P, Català-Moll F. Activation-induced cytidine deaminase targets SUV4-20-mediated histone H4K20 trimethylation to class-switch recombination sites. Sci Rep. 7(1), 7594 (2017). 4. Aguilera A, García-Muse T. R loops: from transcription byproducts to threats to genome stability. Mol Cell. 46(2), 115-124 (2012). 5. Halász L, Karányi Z, Boros-Oláh B. RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases. Genome Res. 27(6), 1063-1073 (2017). 6. Ginno PA, Lott PL, Christensen HC, Korf I, Chédin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol Cell. 45(6), 814-825 (2012). 7. Roy D, Zhang Z, Lu Z, Hsieh CL, Lieber MR. Competition between the RNA transcript and the nontemplate DNA strand during R-loop formation in vitro: a nick can serve as a strong R-loop initiation site. Mol Cell Biol. 30, 146-159 (2010). 8. Roy D, Lieber MR. G clustering is important for the initiation of transcription induced R-loops in vitro, whereas high G density without clustering is sufficient thereafter. Mol Cell Biol. 29, 3124–3133 (2009). 9. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337, 816-821 (2012). 10. Pavri R. R Loops in the Regulation of Antibody Gene Diversification. Genes (Basel). 8(6), 154 (2017). 11. Skourti-Stathaki K, Proudfoot NJ. A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression. Genes Dev. 28(13),1384-1396 (2014). 12. Richard P, Manley JL. R Loops and Links to Human Disease. J Mol Biol. 429(21),3168-3180 (2017). 13. Pearson CE, Nichol Edamura K, Cleary JD. Repeat instability: mechanisms of dynamic mutations. Nat Rev Genet. 6(10), 729-42 (2005). 14. Moreira MC, Klur S, Watanabe M. Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2. Nat Genet. 36(3), 225-227 (2004). 15. Salvi JS, Mekhail K. R-loops highlight the nucleus in ALS. Nucleus. 6(1), 23-29 (2015). 16. Ribeiro de Almeida C, Dhir S, Dhir A. RNA Helicase DDX1 Converts RNA GQuadruplex Structures into R-Loops to Promote IgH Class Switch Recombination. Mol Cell. 70(4), 650-662 (2018). 17. Song C, Hotz-Wagenblatt A, Voit R, Grummt I. SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability. Genes Dev. 31(13), 1370-1381 (2017). 18. Domínguez-Sánchez MS, Barroso S, Gómez-González B, Luna R, Aguilera A. Genome instability and transcription elongation impairment in human cells depleted of THO/TREX. PLoS Genet. 7(12), e1002386 (2011). 19. Sarkies P, Murat P, Phillips LG, Patel KJ, Balasubramanian S, Sale JE. FANCJ coordinates two pathways that maintain epigenetic stability at G-quadruplex DNA. Nucleic Acids Res. 40(4), 1485-1498 (2012). 20. van Wietmarschen N, Merzouk S, Halsema N, Spierings DCJ, Guryev V, Lansdorp PM. BLM helicase suppresses recombination at G-quadruplex motifs in transcribed genes. Nat Commun. 9(1), 271 (2018). 21. Crossley MP, Bocek M, Cimprich KA. R-Loops as Cellular Regulators and Genomic Threats. Mol Cell. 73(3), 398-411 (2019). 22. Chaudhuri J, Alt FW. Class-switch recombination: interplay of transcription, DNA deamination and DNA repair. Nat Rev Immunol. 4(7), 541-552 (2004). 23. Chi X, Li Y, Qiu X. V(D)J recombination, somatic hypermutation and class switch recombination of immunoglobulins: mechanism and regulation. Immunology. 10.1111/imm.13176 (2020). 24. Andreassen PR, Ren K. Fanconi anemia proteins, DNA interstrand crosslink repair pathways, and cancer therapy. Curr Cancer Drug Targets. 9(1), 101-117 (2009). 25. Niraj J, Färkkilä A, D'Andrea AD. The Fanconi Anemia Pathway in Cancer. Annu Rev Cancer Biol. 3, 457-478 (2019). 26. Palovcak A, Liu W, Yuan F, Zhang Y. Maintenance of genome stability by Fanconi anemia proteins. Cell Biosci. 7, 8 (2017). 27. Renaud E, Barascu A, Rosselli F. Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells. Nucleic Acids Res. 44(2), 648-56 (2016). 28. Murina O, von Aesch C, Karakus U, Ferretti LP, Bolck HA, Hänggi K, Sartori AA. FANCD2 and CtIP cooperate to repair DNA interstrand crosslinks. Cell Rep. 7(4), 1030 (2014). 29. Okamoto Y, Iwasaki WM, Kugou K. Replication stress induces accumulation of FANCD2 at central region of large fragile genes. Nucleic Acids Res. 46(6), 2932- 2944 (2018). 30. Okamoto Y, Abe M, Itaya A. FANCD2 protects genome stability by recruiting RNA processing enzymes to resolve R-loops during mild replication stress. FEBS J. 286(1), 139-150 (2019). 31. Liang Z, Liang F, Teng Y. Binding of FANCI-FANCD2 Complex to RNA and RLoops Stimulates Robust FANCD2 Monoubiquitination. Cell Rep. 26(3), 564- 572.e5 (2019). 32. Nakamura M, Kondo S, Sugai M, Nazarea M, Imamura S, Honjo T. High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells. Int Immunol. 8(2), 193-201 (1996). 33. Nguyen TV, Riou L, Aoufouchi S, Rosselli F. Fanca deficiency reduces A/T transitions in somatic hypermutation and alters class switch recombination junctions in mouse B cells. J Exp Med. 211(6), 1011-1018 (2014). 34. Pan X, Chen Y, Biju B. FANCM suppresses DNA replication stress at ALT telomeres by disrupting TERRA R-loops. Sci Rep 9, 19110 (2019). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70261 | - |
| dc.description.abstract | B 細胞經由輔助型T 細胞 (T helper cell) 活化後會進行體細胞超突變 (somatic hyper-mutation, SHM) 以及免疫球蛋白類型轉換 (class switch recombination, CSR)。 體細胞超突變 (somatic hyper-mutation, SHM) 會在免疫球蛋白重鏈及輕鏈 (immunoglobulin heavy chain and light chain) 的可變區 (variable region) 產生高機率的點突變，增加抗體的多樣性進而對抗原的親和力產生影響，而對抗原親和力較強的抗體會逐漸增加，此為抗體親和力成熟過程。免疫球蛋白類型轉換 (class switch recombination, CSR) 會改變免疫球蛋白重鏈 (immunoglobulin heavy chain) 的恆定區 (constant region) ，但可變區 (variable region) 保持不變，因此抗體對抗原的親和力不會改變，而改變抗體的恆定區 (constant region) 會使B 細胞初始表現的IgM 及IgD 轉換成IgG、IgE 或IgA 等其他不同種類的抗體，而在這個轉換的過程中，在免疫球蛋白重鏈 (immunoglobulin heavy chain) 上的轉換區 (switch region) 會產生R-loop。R-loop 是由RNA:DNA hybrid 及single-stranded DNA 所組成的結構，然而，在細胞中過多的R-loop 累積會造成DNA 雙股斷裂，進而影響基因的穩定性，因此，R-loop 需要被嚴格的調控。在本篇論文中，我們致力於找尋在 免疫球蛋白類型轉換 (class switch recombination, CSR) 中調控R-loop 的因子，其中，我們發現Fancd2 (Fanconi anemia group D2) 可能參與調控免疫球蛋白類型轉換 (class switch recombination, CSR) 中的R-loop 形成過程進而影響抗體的多樣性。 | zh_TW |
| dc.description.abstract | Immunoglobulin rearrangements include somatic hyper-mutation (SHM) and class switch recombination (CSR). Both of them are important for antibody diversification, a process to help fighting against and protecting cells from all types of environmental antigens and threats. The R-loop is a three-stranded structure of nucleic acids consisting of a RNA:DNA hybrid and displaced single-stranded DNA (ssDNA). It is involved in various biological processes, such as recruitment of chromatin regulators, heterochromatin formation, and transcription termination. On the other hand, excess of R-loops formation would cause DNA break, replication fork stalling, as well as stalling and collisions between transcription and replication complexes. All of the above might lead to higher genome instability and evolution rate, thus R-loop formation needs to be strictly constrained and regulated. Here, we focus on searching for key R-loop regulating factors during the process of class switch recombination (CSR) at DNA encoded for the immunoglobulin heavy-chain (IgH), an essential process for B cell maturation. When B cells receive cytokine stimuli, R-loops form at switch region leading to DNA deletion and replacement of downstream DNA with another constant region. In our studies, we first found that Fancd2, a repair factor whose mutations are genetically linked to Fanconi anemia, might play an important regulatory role on R-loop formation, subsequently CSR process and antibody diversification. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:24:56Z (GMT). No. of bitstreams: 1 U0001-1508202014293800.pdf: 5946006 bytes, checksum: 1b7400839e6c8f1548dbe47f01033506 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 致謝 ...i 中文摘要 ...ii ABSTRACT ...iii INTRODUCTION ...1 1. R-loop ...1 1.1 R-loop formation ...1 1.2 Physiological role of R-loop ...2 1.3 R-loop-mediated genome instability and related diseases ...4 1.4 R-loop regulatory factors ...5 1.5 R-loop-mediated class switch recombination in B cells ...6 2. Fanconi anemia ...7 2.1 Fanconi anemia pathway and interstrand crosslink repair ...7 2.2 Fanconi anemia pathway and R-loop ...9 SPECIFIC AIM ...10 MATERIALS AND METHODS ...11 RESULTS ...18 1. Confirmation of CH12F3-2A cell line as a desired model to investigate R-loop formation associated with class switch recombination ...18 1.1 CIT induce CSR in CH12F3-2A cells ...18 1.2 CIT stimulate R-loop formation over IgH switch (S) regions in CH12F3-2A cells ...19 2. Fancd2 regulates R-loop formation over IgH S regions and further impairs CSR ...20 2.1 Fancd2 binds to IgH S regions ...20 2.2 Fancd2 impedes CSR ...21 2.3 Recruitment of Fancd2 to IgH S regions declines in Fancd2-depleted CH12F3-2A cells ...22 2.4 Fancd2 inhibits R-loop formation at IgH S regions ...22 3. Possibility of participation by other factors from Fanconi anemia pathway in CSR ...23 3.1 CSR is not regulated by Fanca ...24 3.2 Fancm is a possible factor to control CSR ...25 3.3 Fancd2 regulates CSR dependently of Fanci ...26 DISCUSSION ...27 TABLES AND FIGURES ...29 Table 1. RNAi Lentivirus ...29 Table 2. List of primers ...30 Figure 1. CSR is induced after stimulated with CIT for 72 hours in CH12F3-2A cells ...31 Figure 2. R-loop levels over IgH switch (S) regions increase after stimulated with CIT for 24 hours in CH12F3-2A cells ...32 Figure 3. Fancd2 binds to IgH S regions after stimulated with CIT for 24 hours in CH12F3-2A cells ...34 Figure 4. Fancd2 protein expression decreases in Fancd2-depleted CH12F3-2A cells ...35 Figure 5. Fancd2 knockdown promotes CSR in CH12F3-2A cells ...36 Figure 6. Fancd2 binding to IgH S regions decreases in Fancd2-depleted CH12F3-2A cells ...38 Figure 7. R-loop levels over IgH S regions increase in Fancd2-depleted CH12F3-2A cells ...41 Figure 8. Fanca knockdown has no effect on CSR in CH12F3-2A cells ...46 Figure 9. Fancm knockdown may facilitate CSR in CH12F3-2A cells ...49 Figure 10. Fanci knockdown affects CSR in CH12F3-2A cells ...52 SUPPLEMENTAL INFORMATION ...55 Figure S1. Summary of the switch efficiency of CSR after stimulated with CIT alone or different combination of CIT for 72 hours in CH12F3-2A cells ...55 Figure S2. Anti-CD40, IL-4 or TGF-β deficiency abolishes CSR in Fancd2-depleted CH12F3-2A cells ...58 REFERENCES ...62 | |
| dc.language.iso | en | |
| dc.subject | 體細胞超突變 | zh_TW |
| dc.subject | B 細胞 | zh_TW |
| dc.subject | 免疫球蛋白類型轉換 | zh_TW |
| dc.subject | 抗體 | zh_TW |
| dc.subject | R-loop | zh_TW |
| dc.subject | Fancd2 | zh_TW |
| dc.subject | antibody | en |
| dc.subject | Fancd2 | en |
| dc.subject | B cell | en |
| dc.subject | somatic hyper-mutation | en |
| dc.subject | class switch recombination | en |
| dc.subject | R-loop | en |
| dc.title | 探討修復因子Fancd2在R-loop形成及免疫球蛋白類型轉換的調控機制 | zh_TW |
| dc.title | Investigate on the regulatory mechanisms of repair factor Fancd2 on R-loop formation and immunoglobulin class switch recombination | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 109-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林敬哲(Jing-Jer Lin),黃楓婷(Feng-Ting Huang) | |
| dc.subject.keyword | B 細胞,體細胞超突變,免疫球蛋白類型轉換,抗體,R-loop,Fancd2, | zh_TW |
| dc.subject.keyword | B cell,somatic hyper-mutation,class switch recombination,antibody,R-loop,Fancd2, | en |
| dc.relation.page | 67 | |
| dc.identifier.doi | 10.6342/NTU202003515 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-11-20 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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